Fumonisin Production by Fusarium verticillioides

J. Agric. Food Chem. 2005, 53, 8565−8571

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Fumonisin Production by Fusarium verticillioides Strains Isolated from Maize in Mexico and Development of a Polymerase Chain Reaction to Detect Potential Toxigenic Strains in Grains DIANA SAÄ NCHEZ-RANGEL, ANDREA SANJUAN-BADILLO,

AND JAVIER

PLASENCIA*

Departamento de Bioquı´mica, Facultad de Quı´mica, Universidad Nacional Auto´noma de Me´xico Av. Universidad y Copilco, 04510 Me´xico, D.F., Me´xico

Fumonisins are mycotoxins produced by Fusarium verticillioides (Sacc. Nirenberg) in maize (Zea mays L.), a staple crop in Mexico. In this study, we report the isolation and identification of 67 Fusarium strains isolated from maize kernels collected in Northwest and Central Mexico. The strains were characterized regarding fumonisin B1 production and the presence of the FUM1 gene. F. verticillioides was the predominant species isolated in both geographic regions, but the isolates from Northwest Mexico produced higher levels of fumonisin. A polymerase chain reaction (PCR)-based method, to detect a region of the FUM1 gene involved in fumonisin biosynthesis, was developed and employed to detect mycotoxigenic fungi in pure culture and in contaminated maize. The presence of the FUM1 gene was associated with fumonisin production in most isolates, except seven that did not synthesize fumonisin but contained the gene in their genome. The PCR method allowed the direct detection of fungal contamination in ground corn and could be employed to screen for the presence of potential mycotoxigenic fusaria. KEYWORDS: Mycotoxin; fumonisin B1; Fusarium verticillioides; polymerase chain reaction; maize

INTRODUCTION

Fumonisins are sphingoid-like mycotoxins produced by Fusarium Verticillioides (Sacc. Nirenberg, syn. moniliforme Sheldon), formerly known as Fusarium moniliforme, that have been reported worldwide in maize and in other agricultural products. Although other Fusarium spp. such as Fusarium proliferatum, Fusarium napiforme, and Fusarium nygamai (1) produce fumonisins as well, F. Verticillioides is the most common species isolated from corn (2, 3). This fungus causes ear and stalk rot and has been associated with all of the developmental stages in the maize plant (4), as it is seed borne and can be transmitted from seed to seedling, colonizing the developing plant systemically without producing any conspicuous symptoms, until reaching the kernel. Ear and kernel rots are favored by warm, dry weather during the grain-filling period (3), causing major damage and losses. Even if the rot is not visible, the fungus might be growing and producing fumonisins and other toxins, reducing grain value. Fumonisin B1 is the most abundant toxin produced by most F. Verticillioides isolates and accounts for 70-95% of the total fumonisins (5). Natural incidence of this mycotoxin is a health concern because it has been demonstrated to cause leukoencephalomalacia in equines, pulmonary edema in porcines, and has been included in group 2B (possibly carcinogenic in humans) by the International * To whom correspondence should be addressed. Tel: 52-55-56225275. Fax: 52-55-56225329. E-mail: [email protected].

Agency for Research on Cancer (6). Although there is no direct evidence on human toxicity, epidemiological studies have shown various degrees of association between high incidence of human esophageal cancer (7, 8) and F. Verticillioides or fumonisin in the presence in maize. Therefore, recommendations for maximum levels of fumonisin in foods and feeds have been issued by the Food and Drug Administration (9). Risk assessment studies have been carried out in several countries considering the average contamination level of sampled products and daily consumption of maize. In The Netherlands, for example, a hypothetical daily intake of 1 µg of fumonisin has been suggested as tolerable, considering that only 8 g of corn are consumed per day per person (10). This value is rather low as compared to other countries, like Mexico, where the average consumption of maize is estimated between 222 and 325 g/per day per person (11). Most of the maize consumed in Mexico is in the form of tortillas, which are baked from alkaline-cooked corn kernels in a process called nixtamalization (12). Fumonisin B1 has been detected in tortillas acquired in Mexico in levels ranging from 0.2 to 1.8 µg/g (13). However, this process might eliminate up to 70% of the initial fumonisin content and generates a hydrolyzed fumonisin (14), although the degree of removal depends on nixtamalization conditions. The hydrolyzed product has been detected in levels ranging from 0.01 to 0.1 µg/g in tortillas (15) and might be responsible for the toxicity shown in rats fed with nixtamalized contaminated corn (16).

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Animal toxicoses, probably due to fumonisin contamination of corn, have also been described in Mexico. In a documented outbreak of equine leukoencephalomalacia in the southwestern state of Oaxaca, where over 100 donkeys died, postmortem examination of three of them revealed macroscopic and microscopic cerebral white matter liquefactive necrosis. Fumonisin B1 was detected in levels ranging from 0.6 to 28.5 µg/g in 12 out of 14 corn and feed samples taken from the area (17), but no fungal isolation was performed. In a systematic 2 year survey carried out in Northwest Mexico in 1999 and 2000, 190 corn samples were collected from the field and from storage facilities and analyzed for fumonisin levels and Fusarium spp. presence. The samples yield average fumonisin B1 contents of 1.5 µg/g in the first year and 2.6 µg/g in the second year, as well as a high incidence (70%) of F. moniliforme (18). A more limited survey was carried out in Northeast Mexico, where 34 F. moniliforme strains were isolated and 33 of them produced fumonisins in cracked maize (19). Vegetative compatibility tests identified all of these isolates as the Gibberella fujikuroi mating population A, which is known to produce high fumonisin levels (2). Maize is grown all over Mexico with very distinct environmental and production conditions. In Central and Southern Mexico, mostly open-pollinated landraces and locally adapted hybrids are grown in rain fed, low-input agriculture. In contrast, hybrid seed is employed with high pesticide and fertilizer input and frequently under irrigation in Northwest Mexico (20). Associated with this high variability in maize genotypes and growing conditions, F. Verticillioides populations would be expected to show high variability as well in several phenotypic characteristics including fumonisin B1 production. Studies on fumonisin production potential of a given strain typically include isolation of F. Verticillioides in a selective media and then obtaining single-spore isolates for identification based on morphological characters (21). Such isolates are inoculated in a suitable substrate for fumonisin production such as cracked corn or rice grain, and the fumonisin produced is analyzed by qualitative and quantitative methods (1, 19). In this work, we report a highly specific polymerase chain reaction (PCR) method using primers on the β-cetoacyl reductase domain of the polyketide synthase enzyme (FUM1 gene) that was able to distinguish fumonisin-producing strains from nonproducing strains and species. MATERIALS AND METHODS Maize Samples. Maize samples were either collected in the field or directly with the grower in local markets. The collection site in Northwest Mexico was circumscribed to the Yaqui Valley, which is an important agricultural area in the state of Sonora. Samples were obtained in 2000 and consisted mainly of hybrid maize. Maize samples from Central Mexico were collected in 1999 and 2000 and mainly came from the State of Mexico and represented locally adapted landraces. Isolation and Identification of Fusarium spp. The Fusarium sp. was isolated from both moldy and asymptomatic kernels. Briefly, kernels were surface disinfected by immersion in a 0.5% NaClO solution for 2 min, decanted, and washed with sterile water for another 2 min. The washing was repeated twice, and the seeds were air-dried on a sterile filter paper. Two selective media were employed for Fusarium spp. isolation, pentachloronitrobenzene (PCNB) agar (21) and benzoxazolinone agar (BOA). The latter has been reported as highly selective for F. Verticillioides isolation (22). Up to 18 seeds were placed on each plate containing the selective media and incubated for 5 days at room temperature under fluorescent light. Fungal colonies were transferred to PDA agar and incubated under the same conditions for 5-7 days. Single-spore isolates were obtained by preparing a spore suspension of the PDA culture in 10 mL of sterile water and streaking

Sa´nchez-Rangel et al. a loophole on water agar according to Nelson et al. (21). Species identification was performed using the morphological criteria described by Nelson et al. (21). All single-spored strains were preserved in sterile soil according to Windels et al. (23). Fumonisin B1 Production and Analysis. Potential fumonisin production for each strain was evaluated by inoculating autoclaved rice medium with each single-spore isolate according to Abbas et al. (24). Briefly, 15 g of long-grain parboiled rice was mixed with 9 mL of distilled water in a 50 mL flask and allowed to stand for 1 h at room temperature. The flasks were autoclaved for 1 h at 121 °C twice with a 24 h interval. Once the media was cooled, it was inoculated with each isolate and incubated for 4 weeks at room temperature (24-27 °C) in the dark. At the end of the incubation period, the moldy grain was collected and dried in an incubator at 47-50 °C. A small-scale method was adapted for rapid fumonisin extraction from the dry grain. A subsample of 0.5 g was transferred to a 15 mL Falcon tube containing 5 mL of acetonitrile-water (1:1; v/v). The tubes were kept at room temperature for 12 h, placed in an orbit shaker at 200 rpm for 1 h, and centrifuged for 20 min at 2000g in a Sorvall RT6000D benchtop centrifuge, and the supernatant was recovered for analysis. A preliminary qualitative analysis of fumonisin B1 content was performed by thin-layer chromatography (TLC) on silica gel plates employing ethyl acetate-acetic acid-water (6:3:1; v/v/v) as the mobile phase and visualized with a 0.5% p-anisaldehyde solution (25). Rf values of the spot were compared with a commercial standard of fumonisin B1 (Sigma Chemical Co., St. Louis, MO). If a spot was detected on the crude extract, it was analyzed directly by high-performance liquid chromatography (HPLC); otherwise, it was cleaned-up and concentrated as described below for contaminated maize samples. HPLC analysis of fumonisins was performed according to Sydenham et al. (26) employing a Shimadzu LC-10AD delivery module coupled to a Shimadzu RF-10AXL fluorescence detector (Shimadzu Corporation, Kyoto, Japan) set at 335 nm excitation/440 nm emission wavelengths. Fumonisins were derivatized with o-phthaldialdehyde (Sigma Chemical Co.) reagent [2.5 mg OPA dissolved in 50 µL of ethanol and 2.5 µL of 2-mercapthoethanol in 2.45 mL of 3% borate buffer, pH 10.5] and separated on a 15 cm × 4.6 mm i.d., 5 µm, SuperCosil LC-18 analytical C18 column (Supelco, St. Louis, MO) under isocratic conditions with a flow rate of 1.5 mL/min. The mobile phase employed consisted of methanol-0.1 M NaH2PO4 in a 68:32 v/v ratio, pH 3.3. Purified fumonisin B1 (Sigma Chemical Co.) was employed to prepare a stock solution (1 mg/mL) in 1:1 acetonitrile-water and diluted to obtain standard solutions with decreasing concentrations. These solutions were employed to construct calibration curves (3, 12, 24, and 50 ng/mL). Primer Design. Two sets of primers were employed in this study. A set of universal primers for fungal DNA consisted of primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′), which amplified a 600 bp DNA fragment containing the internal transcribed spacer (ITS) sequences and the 5.8S ribosomal RNA gene (27). These primers are based on the sequences of the 28S and 18S ribosomal RNA genes that show a high degree of conservation. For the detection of fumonisin-producing strains, specific primers were designed using the Primer3 software (www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) based on the sequence of the F. Verticillioides FUM1 gene (AF155773) reported by Proctor et al. (28). Several primer combinations were tested, and a primer set [FUM53F (5′-CTTGAACGCGGAGCTAGATTAT-3′) and FUM53R (5′-ATCCGTGTATGCATATGTCGAG-3′)] based on the sequence of the exon 3 that amplifies a 354 bp fragment was employed in a PCR using 50-100 ng of fungal DNA. DNA Extraction and Purification. Each strain was grown on PDA in 10 cm diameter Petri dishes for 9-10 days at room temperature under fluorescent light. A volume of 10 mL of 0.5% Tween-20 solution was added to each plate and shaken at 75-100 rpm for 2 h at room temperature. The spore and mycelia suspension was collected and transferred to a 25 mL centrifuge tube and centrifuged for 20 min at 600g in a Jouan MR1812 refrigerated centrifuge. The supernatant was discarded, and the pellet was washed with 10 mL of sterile water and centrifuged again under the same conditions. The pellet was resuspended in 1 mL of sterile water, transferred into a 2 mL Eppendorf tube, and

Detection of Fumonisin-Producing Fusarium in Maize centrifuged for 10 min at 10000g. The pellet was frozen in liquid nitrogen, and 1 mL of DNAzol (Invitrogen, Carlsbad, CA) was added to the tube and incubated at room temperature until thawed. Then the tube was centrifuged at 10000g for 10 min at room temperature, and the supernatant containing the DNA was transferred to a fresh tube containing 0.5 mL of 2-propanol (-20 °C), which was mixed by inversion and incubated at room temperature for 3 min before a third centrifugation step (10 min at 7000g). The supernatant was discarded, and the pellet was washed with 1 mL of 70% ethanol. The pellet was air-dried for 15 min and dissolved in 20 µL of 8 mM NaOH and incubated for 15 min at 37 °C, then centrifuged at 7000g to eliminate the insoluble material. The supernatant neutralized by adding 1 M TrisHCl, pH 8.0, so this solution had approximately a final concentration of 5%. PCR. PCR for amplification of the FUM1 DNA fragment was carried out in an Applied Biosystems GeneAmp System 9700 thermocycler (Applied Biosystems, Foster City, CA) programmed for one cycle of 3 min at 94 °C, 27 cycles of 40 s at 94 °C, 40 s at 56 °C, 40 s at 72 °C, and one cycle of 7 min at 72 °C. Reactions were carried out in volumes of 20 µL containing 50-100 ng of template DNA in 1 µL, 0.3 µM each primer (20 µM), 0.02 of platinum Taq DNA polymerase, 2 µL of 10X PCR buffer, 2 µL of MgCl2 (25 mM), and 0.4 µL of dNTPs (10 mM) mix. Amplification of the ITS region was performed with the same temperature program as described above, but with 30 cycles, the primer concentration was adjusted to 0.5 µM. PCR products were resolved in a 1.5% agarose ethidium bromide gel in 40 mM Tris acetate and 2 mM EDTA buffer. The 100 bp DNA ladder (Promega Corp., Madison, WI) was used as a molecular size marker. DNA was purified from 1 week old cultures of Fusarium sp. and employed for PCR procedures. To guarantee that the purified DNA was an adequate substrate for amplification and free of inhibitors, all DNAs were tested first with the generic primers ITS4 and ITS5, all of them yielding a 600 bp fragment (Sa´nchez-Rangel, results not shown). Analysis of Contaminated Samples. Fifty grams of cracked maize kernels was placed in a 250 mL Erlenmeyer flask and moistened with 15 mL of deionized water and kept for 1 h at room temperature. The flasks were autoclaved for 1 h at 121 °C twice with a 24 h interval. Once the media was cooled, it was inoculated with strain MY6, a high fumonisin producer, and a second flask with strain MZ3-1 that did not produce fumonisin B1. The flasks were incubated for 4 weeks at room temperature (24-27 °C) in the dark. The moldy grain was then collected and dried in an incubator at 47-50 °C. A 20 g subsample of this maize was ground to a fine powder in a grinding mill (Bel-Art products, Pequannock, NJ). This flour was blended at different ratios (10, 1%; 0.1, 0.01, 0.001, and 0.0001%; w/w) with noncontaminated ground corn for fumonisin and DNA extractions according to the following protocols. Samples were extracted and cleaned according to Hopmans and Murphy (29). Briefly, 10 g of the ground material was placed in a 250 mL flask with 50 mL of acetonitrile-water (1:1; v/v) and shaken at 200 rpm for 30 min at room temperature. The extract was filtered through a Whatman 4 filter paper, and a 1 mL aliquot was collected and passed through a conditioned SPE C18 Sep-Pak column (Waters, Milford, MA). Fumonisin was eluted with 1 mL of acetonitrile-water (70:30 v/v), and the eluate was analyzed by TLC and HPLC as described above. DNA was extracted from 100 mg of the ground flour in a 2 mL Eppendorf tube containing 0.5 mL of 25 mM Tris and 50 mM EDTA solution, pH 7.5. A volume of 7.5 µL of lyticase (20 mg/mL) was added to the tube that was placed in a shaker for 1 h at room temperature. The tube was centrifuged at 10000g for 10 min in a MC12 Sorvall microcentrifuge, and the pellet was immersed in liquid nitrogen for 3 min, 1 mL of DNAzol was added, and the protocol was completed as described above. The same procedures were performed with naturally contaminated maize samples that were ground to detect fumonisin B1 and the DNA of fumonisin-producing fungi. RESULTS

In this work, 67 Fusarium strains were isolated and observed for identification. Fifty-four isolates (80.6%) were identified as F. Verticillioides based on morphological characters, and a few

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other species of low incidence were identified as well: Fusarium aVenaceum (four isolates), Fusarium niVale (two isolates), Fusarium oxysporum (two isolates), Fusarium subglutinans (two isolates), and Fusarium chlamydosporium, Fusarium semitectum, and Fusarium sporotrichioides with one isolate each (Table 1). F. Verticillioides was recovered with high frequency, either from kernel or ear samples by using both PCNB agar and BOA agar. Fumonisin production was evaluated in all strains using a natural substrate medium that allows the synthesis of the mycotoxin, showing a wide variation in this parameter. By using the criteria of Nelson (30), 11 out of 13 strains isolated from the Northwest produced high levels (above 500 µg/g), one intermediate (below 500 µg/g), and one low producer (below 50 µg/g). In contrast, strains isolated from Central Mexico consisted mostly of low producers and nonfumonisin producers and only three intermediate producers. No fumonisin production was detected in any of the other isolated Fusarium species. The specificity of the FUM53F/FUM53R primer set to detect the FUM1 gene was tested with all purified DNA samples. This primer set yielded a unique 354 bp fragment (Figure 1A) in 37 of the 67 DNAs tested. The identity of the amplicon was confirmed by sequencing the cloned DNA fragment obtained from MY6 strain, showing an identical sequence to that reported (AF155773) by Proctor et al. (28). In this 354 bp DNA fragment, an SstI site (GAGCTC) was identified at position 284 (Figure 1B). Upon restriction with this enzyme, two fragments were obtained, a 288 bp DNA fragment and a 66 bp fragment. The former migrated slightly faster than the intact product, thus providing a confirmatory test for all of the amplified products (Figure 1C). Fumonisin production was then associated with the presence of the FUM1 gene (Table 1). The specific PCR amplification product was detected in all 30 strains that produced fumonisin but also in seven F. Verticillioides isolates (PAL1, AZU3, H135-3, MOR2, MZ2-2, JIQ5-5, and CRIB-2) in which the toxin was not detected. All of these strains came from maize samples collected in Central Mexico. To test the feasibility to employ the primers in a diagnostic test that would allow the detection of fumonisin-producing fungi in maize, blends containing known ratios of contaminated maize were analyzed for fumonisin production and the presence of the FUM1 gene. Cracked maize kernels inoculated with strain MY6 contained 3100 µg/g fumonisin B1 and upon 10-fold dilutions with noncontaminated maize, fumonisin B1 levels were reduced down to 0.14 µg/g in the 0.01% blend. The fungal biomass was unmistakably detected by PCR in the inoculated maize and in the first two dilutions, and a fainter band was still detected in the 0.1% blend corresponding to fumonisin levels approximately of 2 µg/g (Figure 2). The same procedure was applied to naturally contaminated kernels from maize ears collected in the Yaqui Valley that contained 32 µg/g fumonisin B1 (Figure 3, lane 4) and showed a strong amplification signal. In contrast, two maize samples collected in Chalco, Edo, Mexico, did not contain fumonisin B1 and were negative for the FUM1 gene (Figure 3, lanes 2 and 3). DISCUSSION

F. Verticillioides is a plant pathogen of maize that occurs with high incidence wherever this crop is grown; however, there are few studies on the incidence of this fungus in maize in Mexico despite the enormous importance of this crop. The use of the species selective BOA media contributed to increase the frequency of isolation of F. Verticillioides, although other species such as F. aVenaceum, F. niVale, F. subglutinans, F. oxysporum,

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Table 1. Fumonisin B1 Production and Presence of the FUM1 Gene in Several Fusarium Species Isolated from Maize in Northwest and Central

Mexico

a

strain

geographic origin

source

species identification

fumonisin B1 production (µg/g)

FUM1 gene

MY1 MY2 MY3 MY4 MY5 MY6 L-189-1 L-189-2 L-189-3 L-176-1 L-176-2 L176-3 L-176-4 PAL1 PAL2 CHQ1 CHQ2 CHQ5 CHQ6 AZU2 AZU3 H135-1 H135-2 H135-3 H135-4 MOR2 MOR3 CRI CRIB-1 CRIB-2 CRIB-3 PALB-1 PALB-2 MORB-1 MORB-2 MORB-3 MEM-1 MEM-2 MEM-3 MEM-4 MCH-1 MCH-2 MCH-3 MCH-4 MCH-5 MEMB-1 MEMB-2 MEMB-3 MEMB-4 MZ1-1 MZ1-2 MZ2-1 MZ2-2 MZ2-3 MZ2-4 MZ3-1 MZ3-4 MZ2B-2 MZ2B-3 MZ3B-3 MZ3B-4 JIQ5-1 JIQ5-2 JIQ5-3 JIQ5-4 JIQ5-5 GUA4-1

Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Yaqui Valley, Son Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Chalco, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Acambay, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Aculco, Edo Mex Jiquipilco, Edo Mex Jiquipilco, Edo Mex Jiquipilco, Edo Mex Jiquipilco, Edo Mex Jiquipilco, Edo Mex Jiquipilco, Edo Mex

ear ear ear ear ear ear ear ear ear ear ear ear ear kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel kernel ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear ear kernel kernel kernel kernel kernel kernel

F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. semitectum F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. subglutinans F. avenaceum F. verticillioides F. subglutinans F. avenaceum F. verticillioides F. chlamidosporium F. verticillioides F. nivale F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. oxysporum F. oxysporum F. nivale F. verticillioides F. avenaceum F. verticillioides F. sporotrichioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. avenaceum F. verticillioides F. verticillioides F. verticillioides F. verticillioides F. verticillioides

1718 1152 1699 1940 1.3 3658 3062 4047 1617 454 1223 1257 1534 NDa ND ND ND ND ND ND ND 174 99 ND 53 ND 0.21 ND ND ND 3.7 1 0.9 1.4 0.9 ND ND ND ND ND ND ND ND ND ND 0.7 ND ND ND ND ND ND ND ND ND ND ND 3.6 0.9 ND ND 0.2